The present invention concerns a novel enzyme, namely aldehyde dehydrogenase (ADH), a process for producing ADH and a process for producing 2-keto-L-gulonic acid (2-KGA) from L-sorbosone utilizing said enzyme. 2-KGA is an important intermediate for the production of vitamin C.
Some microorganisms are known to convert L-sorbosone to 2-KGA. For example, in U.S. Pat. No. 3,907,639, the microorganisms belonging to the genera Acetobacter, Pseudomonas, Escherichia, Serratia, Bacillus, Staphylococcus, Aerobacter, Alcaligenes, Penicillium, Candida and Gluconobacter are reported to be capable of effecting the conversion. Furthermore, Kitamura et al. (Eur. J. Appl. Microbiol., 2, 1, 1975) report that the enzyme oxidizing L-sorbosone found in Gluconobacter melanogenus IFO 3293 requires neither a coenzyme nor an electron acceptor for the development of enzyme activity. Makover et al. (Biotechnol. Bioeng. 17, 1485, 1975) report the presence of L-sorbosone dehydrogenase activity in the particulate fraction of Pseudomonas putida ATCC 21812 and of Gluconobacter melaogenus IFO 3293. They also indicate that neither nicotinamide adenine dinucleotide (NAD) nor nicotinamide adenine dinucleotide phosphate (NADP) acts as a coenzyme for the enzyme. T. Hoshino et al. (Agric. Biol. Chem., 55, 665, 1991) purified and characterized L-sorbosone dehydrogenase from Gluconobacter melanogenus UV10, which requires NAD or NADP as a coenzyme.
In the context of the present invention, microorganisms belonging to the genus Gluconobacter have been studied more closely and, as a result, it has been found that the further novel ADH which catalyzes the oxidation of L-sorbosone to 2-KGA can be obtained from said microorganisms. Furthermore, it has been found that the purified ADH provided by the present invention oxidizes L-sorbosone to 2-KGA in the presence of electron acceptors, such as 2,6-dichlorophenolindophenol (DCIP) and phenazine methosulfate (PMS), ferricyanide or cytochrome c, but that NAD, NADP and oxygen are not suitable as electron acceptors. Thus, the ADH provided by the present invention is clearly distinct from the known L-sorbosone dehydrogenase.